Defining the impact of glycan-microdomains of the HIV-1 Env glycan shield

摘要 人类免疫缺陷病毒1（HIV-1）是全球主要病原体，2019年新增感染170万例。 HIV-1包膜糖蛋白（Env）是唯一的病毒表面抗原。Env，gp 120/gp 41二聚体的三聚体 亚基被N-聚糖密集糖基化。对于gp 120亚基，N-聚糖占其总糖链的50%以上。 分子量，并形成一个保护表面，所谓的“聚糖盾”，与细胞受体相互作用 和免疫系统，并保护病毒免受中和抗体（nAb）的侵害。最近， 基于免疫逃逸变体的序列分析， 已经失去了一些潜在的N-糖基化位点（NGS），并获得了其他位点。尽管取得了重大进展， 尽管对HIV-1 Env的生物学了解不多，但在分子水平上N-聚糖的复杂性和功能仍然很差。 明白基于我们的生物信息学和实验研究，我们最近提出Env聚糖 屏蔽可以被定义和分析为不同结构聚糖微区。使用天然存在的Env 在进化枝B病毒的变异中，我们分析了高度保守的N262聚糖周围的聚糖， 称为高甘露糖斑块（HMP），靠近Env三聚体的顶点。这些HMP聚糖由不同的 在N262附近的4至6个NGS的组合似乎形成聚糖工作单元。基于我们 根据已发表的结果和初步数据，我们假设Env外结构域，gp 120聚糖盾， 可以定义为不同结构聚糖微域的结构组分。我们建议 通过评估HIV-1及其免疫逃逸变体的结构和功能， 本文描述了包含Env聚糖微结构域的一系列N-聚糖组合的功能影响。具体地说， 我们建议确定N262锚定的HMP微结构域的不同NGS组合如何跨越 变体和进化枝影响Env功能（目的1），以探测不同N262-聚糖的结构排列 在功能性重组Env三聚体的背景下对NGS组合进行聚类（Aim 2），并评估 HIV-1 Env聚糖盾的其他潜在聚糖簇的功能影响（目标3）。完成这些 目标，我们将使用功能，生物分析，结构生物信息学和分子动力学的组合 模拟方法。这些研究的结果将检验我们提出的关于功能影响的假设 的结构组成部分的聚糖盾牌，聚糖微域，并将决定如何变化 这些微区的组成影响聚糖屏蔽的功能。这些数据将推动我们 了解HIV-1的分子生物学以及影响病毒传播和持久性的因素。 最终，这些研究将产生关于Env漏洞的新信息，这些漏洞可以被利用来开发 未来的治疗方法。

ABSTRACT Human immunodeficiency virus 1 (HIV-1) is a major global pathogen, with 1.7 million new infections in 2019. The HIV-1 envelope glycoprotein (Env) is the sole virus-surface antigen. Env, a trimer of gp120/gp41 dimer subunits, is densely glycosylated by N-glycans. For the gp120 subunit, N-glycans contribute over 50% of its molecular mass and form a protective surface, the so-called “glycan shield”, that interacts with cellular receptors and the immune system and protects the virus from neutralizing antibodies (nAbs). Recently, the concept of an “evolving glycan shield” has been proposed, based on the sequence analysis of immune-escape variants that have lost some potential N-glycosylation sites (NGS) and gained others. Despite significant strides in understanding the biology of HIV-1 Env, the complexity and function of N-glycans at a molecular level are poorly understood. Based on our bioinformatics and experimental studies, we recently proposed that the Env glycan shield may be defined, and analyzed, as distinct structural glycan-microdomains. Using naturally occurring Env variants of a clade B virus, we analyzed the glycans surrounding the highly conserved N262 glycan, in the so- called high-mannose patch (HMP) near the apex of the Env trimer. These HMP glycans, comprised of different combinations of 4 to 6 NGS in the proximity of N262, appear to form a glycan working unit. Based on our published results and preliminary data, we hypothesize that Env outer domain, gp120 glycan shield, has structural components that can be defined as distinct structural glycan-microdomains. We propose to test our hypothesis in different clades of HIV-1 and their immune-escape variants by assessing structural and functional impacts of a range of N-glycan combinations that comprise Env glycan-microdomains. Specifically, we propose to determine how different NGS combinations of the N262-anchored HMP-microdomain across variants and clades influence Env functions (Aim 1), to probe the structural arrangement of different N262-glycan cluster NGS combinations in the context of functional recombinant Env trimers (Aim 2), and to assess the functional impacts of other potential glycan-clusters of the HIV-1 Env glycan shield (Aim 3). To accomplish these goals, we will use a combination of functional, bioanalytical, structural bioinformatics, and molecular dynamics simulation approaches. The results of these studies will test our proposed hypothesis on the functional impact of the structural components of the glycan shield, glycan-microdomains, and will determine how the varied compositions of these microdomains affect the function of the glycan shield. These data will advance our understanding of the molecular biology of HIV-1 and the factors that impact viral transmission and persistence. Ultimately, these studies will generate new information about Env vulnerabilities that can be exploited to develop future therapeutic approaches.